Live cell imaging systems and methods to validate triggering of immune response

ABSTRACT

Systems and methods for predicting an immune response against a tumor in a patient having the tumor are provided. The relative mass or changes of mass of tumor cells or immune cell in the tumor can be ex vivo observed, and an immune status of the tumor can be determined based on the mass of tumor cells or immune cell. The immune status can provide a guidance to predict the immune response against the tumor in the patient.

This application claims priority to U.S. provisional application withthe Ser. No. 62/435,520, filed Dec. 16, 2016, and which is incorporatedby reference herein.

FIELD OF THE INVENTION

The field of the invention is validation of a neoepitope-based therapyprior to use of a therapeutic composition in a patient.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

Despite recent advances in the diagnosis and treatment of cancer, manytypes of cancers remain difficult to cure or drive into long termremission. Currently, one promising avenue of research is theindividualized therapy of cancers. The concept of individualized therapyis based on the observation that each cancer, in each individual patientor subject, is in some ways unique. Thus, if the therapy is tailored toaddress or target the genetic elements and/or regulatory cellularpathway(s) altered or mis-regulated in a specific tumor, the prospectsof a successful treatment against the tumor are likely to be enhanced.For example, recent development of various immunotherapies is targetedto specifically elicit immune response against cells exposingneoepitopes that are mostly patient-specific and tumor-specific. Whilethe individualized immunotherapy seems promising to treat many tumorsthat could not be effectively treatable previously, the effectiveness ofsuch individualized immunotherapy often unexpectedly vary due tonumerous factors that can reduce the efficacy of the treatment. Forexample, inter alia, some tumor cells may not efficiently process andpresent neoepitope on the cell surfaces, which may lead to a failure ofeliciting immune responses against the tumor cells. In addition, thepatient's specific T-cell population may lack proper responsiveness, forexample, due to checkpoint inhibitory signaling that is prevalent intumor microenvironment, or due to a large fraction of M2 macrophages,regulatory T cells (Tregs), and/or myeloid-derived suppressor cells(MDSCs). Therefore, even where patient and tumor specific neoepitopesare selected or designed for proper MHC presentation, the effectivenessof the individual immunotherapy may vary depending on the tumor celltypes and the tumor microenvironment.

Live cell interferometry (LCI) detects the live cells and provides rapidand real-time quantification of cell mass in cells, thus can be used forvarious assessment including cell mass changes upon changes duringresponse to the drugs. For example, U.S. Patent Pub. No. 2014/0178865discloses measurement of cell mass, or optical cell thickness ofmultiple myeloma cells upon tunicamycin treatment. In another example,U.S. Patent Pub. No. 2016/0103118 discloses identifying T cell receptorsthat respond to specific target cell antigens by detecting mass changesin T cells. However, none of those discloses use of LCI to predictwhether a tumor cell or the tumor mass will be responsive to effectiveimmunotherapy.

Thus, even though various compositions and methods of individualizedimmunotherapy are known in the art, evaluation of individual tumors fortheir suitability for individualized immunotherapy, especially usingLCI, is largely unexplored. Consequently, there is still a need forsystems and methods that help predict effectiveness of individualizedimmunotherapy in a patient.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various methods and systemsof predicting an anticipated immune response to a tumor in a patienthaving the tumor, especially prior to an immunotherapeutic treatment tothe patient, in which the immune status is determined by live cellinterferometry (LCI) analysis of tumor cells and immune competent cellspresent in the tumor. In addition, the LCI analysis of the tumor cellsand immune competent cells can be further extended to evaluate theeffectiveness of the immunotherapeutic treatment.

Thus, in one especially preferred aspect of the inventive subjectmatter, the inventor contemplates a method of predicting an immuneresponse against a tumor in a patient having the tumor. In this method,a tumor sample of the patient is obtained prior to immunotherapeutictreatment of the patient. Then, the mass of a tumor cell and/or the massof an immune competent cell is determined in the tumor sample using livecell interferometry. Using the determined mass of a tumor cell and/orthe mass of an immune competent cell, an immune status of the tumorsample can be further determined, and be used to predict the immuneresponse against the tumor in the patient.

In further contemplated aspects, the step of determining the mass of thetumor and/or immune competent cell will include measuring changes over aperiod of time (e.g., several hours) in the mass of the tumor and/or theimmune competent cell. Additionally or alternatively, the specificinteraction between the tumor cell (isolated or in the tissue) and oneor more types of immune competent cells can be determined, where thetumor cell is located proximally to the immune competent cell, bycomparing the mass of the tumor cell or the immune competent cell withan average mass of other tumor cells or immune competent cells,respectively, in the tumor sample.

It is also contemplated that the methods presented herein may include astep of contacting the tumor sample with an immune stimulatory cytokine,a checkpoint inhibitor, and/or an immunotherapeutic agent (e.g.,neoepitope peptide, nucleic acid encoding a neoepitope, etc.). Wheredesired, the immune competent cell may be an immune competent cell ofthe patient that was previously exposed to an immunotherapeutic agent.

In other aspects, the immune status may be determined to be hot when themass of the tumor cell is substantially smaller than other tumor cellsin the tumor sample, when the mass of the immune competent cell issubstantially larger than other immune competent cells in the tumorsample, and/or when the mass of the immune competent cell issubstantially larger than other immune competent cells in the tumorsample and when the mass of the tumor cell is substantially smaller thanother tumor cells in the tumor sample where the tumor cell is proximalto the immune competent cell. An anticipated immune response may then bedeemed positive when the immune status is determined to be hot. If theimmune status is determined to be cold, it is contemplated thatimmunotherapeutic treatment may be modified or other alternative optionsfor the immunotherapeutic treatment can be provided.

With respect to the tumor sample, it is contemplated that the tumorsample can be a fresh tumor biopsy from the patient or can be anartificial tumor that is generated from a tumor biopsy of the patient.The tumor sample can be further prepared as one or more acute tissueslices, cultured slices, or as a disassociated cell culture (usingadherent or suspended cells, optionally from trypsinated tissue) placedon a dish, or any solid carrier or support. Optionally, the tumor celland the immune competent cell in the tumor sample can be fixed afterlive cell interferometry (LCI) analysis for labeling the cells with oneor more markers and identifying the cell types.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION

Most tumors present as a heterogeneous mass of various types of cells inaddition to the tumor cells, and frequently include various immunecells, bone marrow-derived inflammatory cells, lymphocytes, fibroblasts,as well as an extracellular matrix and epithelial cells structuringsurrounding blood vessels. Proliferation of tumor cells in the tumormicroenvironment typically is closely related to the interaction oftumor cells and other cells surrounding the tumor cells. Especially, thedynamic interactions between the tumor cells and immune cells in thetumor environment often shift balances between a cytotoxic immuneresponse to the tumor and immune-suppression in the tumormicroenvironment such that the tumor cells are in a sensitive status orin a resistant status (e.g., primary resistance, adaptive immuneresistance, acquired resistance, etc.) to a host (e.g., patient) immuneresponse.

The inventors contemplate that the tumor cells in the immune-sensitive(“hot”) status are likely to be susceptible to the immunotherapy andthat the tumor cells in the immune-resistant (“cold”) status are lesslikely to be susceptible to the immunotherapy. Viewed from a differentperspective, the inventors contemplate that effectiveness of theimmunotherapy can be predicted by determining the immune-sensitive orimmune-resistant status of the tumor cells and/or tumormicroenvironment. With that, the inventors have now discovered that theimmune response of a patient against its tumor cells can be predictedsubstantially in real-time (e.g., within less than 24 hours, or withinless than 12 hours, or within less than 6 hours from obtaining tumorcells/tissue) by observing and/or comparing the cell mass of tumor cellsand/or immune competent cells within the tumor mass or tumormicroenvironment. Advantageously, such methods will allow rapidcommencement of immune therapy in a patient upon first biopsy or surgery(and may as such avoid chemotherapy that would otherwise potentiallyharm the immune system).

As used herein, the term “tumor” refers to, and is interchangeably usedwith one or more cancer cells, cancer tissues, malignant tumor cells, ormalignant tumor tissue, that can be placed or found in one or moreanatomical locations in a human body.

As used herein, the term “bind” refers to, and can be interchangeablyused with a term “recognize” and/or “detect”, an interaction between twomolecules with a high affinity with a K_(D) of equal or less than 10⁻⁶M,or equal or less than 10⁻⁷M. As used herein, the term “provide” or“providing” refers to and includes any acts of manufacturing,generating, placing, enabling to use, or making ready to use.

Most preferably, profiling of cell mass of tumor cells and/or immunecompetent cells can be performed using live cell interferometry (LCI),which can determine the cell mass of a plurality of adherent ornon-adherent cells under the microscope almost simultaneously. As usedherein, cell mass is measured using the optical thickness (density) ofthe cell and surrounding environment. Briefly, the optical thickness ofa living cell in a sample can be compared with the optical thickness ofthe surrounding media (e.g., cell culture media, tissue culture media,intermediate buffer, etc.), which is measured by phase retardation ofthe light passing through each cell. The difference between thoseoptical thicknesses is due to the interaction of light with cellularbiomass, which is linearly proportional to the material density of acell. The detailed method and algorithm of the measuring the cell massusing the LCI on a single cell scale is provided in Reed et al.,Biophysical Journal, Volume 101, pp. 1025-1031 (September 2011), 102.3(2012) and Zangle et al., Nature Methods, 11, 1221-1228 (2014), and in amulti-cell scale is provided in U.S. Patent Pub. No. 2014/0178865 toReed, and U.S. Patent Pub. No. 2016/0103118 to Teitell, which are allincorporated by references in their entireties herein.

Optionally, the optical thickness of the cell can be compared and/ornormalized with the physical thickness (e.g., height) of the cell. Anysuitable methods of measuring the physical thickness of cells arecontemplated, including, but not limited to measurement using opticalphase microscope.

The inventors observed that immunologically active cytotoxic orcytolytic immune cells (e.g., active T cell, cytotoxic T cells, activeNK cells, active NKT cells attacking cells upon recognition of theantigens or neoepitopes presented on the cells, etc.) increase theirmass upon interacting with the cells presenting antigens or neoepitopes.Conversely, immunogenic cell (e.g., immune-sensitive tumor cells, tumorcells expressing antigens or neoepitopes) interacting with and beingattacked by the immune cells (e.g., either cytolytic or cytotoxicattack, etc.) are likely to decrease their mass (e.g., due to apoptosis,necrosis, etc.). Therefore, viewed from a different perspective,decreased mass of the immune cells can be an indicator an of inactive orsuppressed immune response or immune system in the tumormicroenvironment, and an increased mass of tumor cells can be anindicative of proliferation of the tumor cells that overcome the immuneresponse against the tumor cells in the tumor microenvironment.

Thus, in one especially preferred embodiment, an immune response againsta tumor in a patient having the tumor can be predicted by determiningthe cell mass of a tumor cell and/or an immune competent cell in a tumorsample. Most typically, the tumor sample is obtained from the patientthrough a biopsy of the tumor tissue during the surgery or regularbiopsy procedure. Preferably, the biopsy tumor tissue can be furtherprocessed for observation under the LCI and other types of microscopicanalysis, if necessary. In one embodiment, the biopsy tumor tissue canbe processed via manual and/or automated tissue slicing (e.g., usingLeica Vibratome, etc.) under semi-sterile conditions, into acutelysliced tissues with a thickness of 100-500 μm, preferably 150-400 μm,and more preferably about 150-300 μm, and most preferably about 200-250μm per each slice. The acutely sliced tumor tissue can be placed on achamber for observation under the LCI and other types of microscopewithin 10 min, within 30 min, or within 1 hour after the slices aregenerated. Additionally, the acutely sliced tumor tissue can be moved toa chamber containing tissue culture media (e.g., Dulbecco's modifiedeagle media (DMEM)-based tissue culture media, Gibco® RPMI-1640-basedmedia, etc.), for example, to be cultured in 37° C., 5% CO2 environmentfor at least 6 hours, at least 12 hours, at least 24 hours, at least 3days, at least 7 days, before the observation under the LCI and othertypes of microscope.

Alternatively, the biopsy tumor tissue or the acutely sliced tumortissue can be processed with enzymatic disassociation using one or moreof trypsin, papain, elastase, hyaluronidase, collagenase, pronase anddeoxyribonuclease, depending on the type, origination, or location ofthe tumor, and optionally in combination with mechanical and chemicaldisassociation tools (e.g., using cation chelators including EDTA andEGTA, etc.). For example, trypsin can be suitably used to disassociatetumor cells originated from brain, epidermis, kidney and lung, andpapain can be suitably used to disassociate tumor cells originated frommuscle. Once disassociated, the cells from the biopsy tumor tissue maybe cultured and maintained in the cell culture medium for at least 6hours, at least 12 hours, at least 24 hours, at least 3 days, at least 7days, before further observation under the LCI and other types ofmicroscope.

In some embodiment, the entire biopsy tumor tissue or the tumor tissueslice can be used. In other embodiments, the biopsy tumor tissue or thetumor tissue slice can be further processed into (e.g., punched, etc.)smaller tissues pieces (e.g., less than 2 mm×2 mm, less than 1.5 mm×1.5mm, less than 3 mm diameter, less than 2 mm diameter, etc.). In suchembodiments, the inventors contemplate that the locations of the smallertumor tissues can be mapped relative to the entire or at least a portionof the biopsy tissue to determine or identify any heterogeneity inimmune-responsiveness status (e.g., immune-sensitive, immune-resistantstatus) among sub-regions of the tumor.

As it represents a tumor microenvironment, the biopsy tumor tissue islikely to include different types of cells including, but not limitedto, tumor cells, immune cells, and epithelial cells (e.g., from theblood vessel surrounding the tumor, etc.). As used herein, immune cellsrefer any cells (cytotoxic or non-cytotoxic immune cells) in the immunesystem, including, but not limited to, B cells, T cells, cytotoxic Tcells, natural killer (NK) cells, natural killer T (NKT) cells,macrophage, monocytes, and innate lymphoid cells. In addition, as usedherein, immune competent cells refer any immune cell that has ability toelicit immune response following the exposure to an antigen. In someembodiments, the immune cells also include myeloid derived suppressorcells (MDSC) that mediate immune suppression in the tumormicroenvironment. In order to mimic the tumor microenvironment in vivo,it is generally preferred that a disassociated cell culture derived froma biopsy tumor tissue includes similar types and ratios of various cellsinteracting with each other. Yet, the inventors also contemplate that insome embodiments, one type of cells can be preferably or dominantlycultured over other types of cells to obtain a more homogeneous group ofcells from the biopsy tumor tissue. For example, the tumor cells fromthe biopsy tumor tissue can be isolated and/or selectively cultured viapreferred culture technique for the tumor cell over other types of cells(e.g., sandwich culture, 3D culture, etc.) or depriving other cell typesfrom the tissue (e.g., enzymatic degradation of stromal cells, etc.).The isolated and/or selectively cultured tumor cells can be furthercultured to form an in vitro artificial tumor mass at least for 2 weeks,at least for 3 weeks, at least for 6 weeks, etc. as will be appreciated,separately cultivated cells can be rejoined (e.g., using same ratio asin tumor tissue) as desired.

The processed or unprocessed tumor tissue (from biopsy tissue) can thenbe analyzed using live cell interferometry to determine the activity ofthe immune cells in the tumor against the tumor cells and/or to identifyimmunogenic or immunologically active cells in the tumormicroenvironment. As noted earlier, it was observed that immunologicallyactive cytotoxic or cytolytic immune cells (e.g., active T cells,cytotoxic T cells, active NK cells, active NKT cells attacking cellsupon recognition of the antigens or neoepitopes presented on the cells,etc.) increase their mass upon interacting with the cells presentingantigens or neoepitopes. Conversely, immunogenic cell (e.g.,immune-sensitive tumor cells, tumor cells expressing antigens orneoepitopes) interacting with and being attacked by the immune cells(e.g., either cytolytic or cytotoxic attack, etc.) are likely todecrease their mass (e.g., due to apoptosis, necrosis, etc.). Therefore,viewed from a different perspective, decreased mass of the immune cellscan be an indicator an of inactive or suppressed immune response orimmune system in the tumor microenvironment, and an increased mass oftumor cells can be an indicative of proliferation of the tumor cellsthat overcome the immune response against the tumor cells in the tumormicroenvironment.

Thus, in a preferred embodiment, at least one tumor cell and/or at leastone immune cell can be selected from the tumor sample (e.g., acuteslices, cultured slices, dissociated cells, unprocessed biopsy tissue)and the mass of those cells can be individually and/or collectivelydetermined using the LCI. Preferably, the selected tumor cell and theimmune cell are proximally located (e.g., within 100 μm, within 50 μm,within 25 μm, within 10 μm, etc.) such that the immune cell and thetumor cell can make a direct or indirect contact and/or interaction witheach other, or at least the molecules secreted or presented by theimmune cell (e.g., cytokines, etc.) can be transmitted to the tumorcells without a significant dilution. Thus, in this embodiment, thetumor cell and the immune cell that can be visibly distinguishable(e.g., by morphology, size, etc.) and located proximally with each othercan be selected to measure the mass. Additionally, the mass of the tumorcell and the immune cell can be compared with other tumor cell and theimmune cell, respectively in the same tissue examined under the LCI orthe limited area of the tissue under the same field of view of the LCI.

It is especially preferred that the immune cells (especially immunecompetent cells) can be visually distinguishable from tumor cells orother types of the cells in the tumor tissue, and also from with eachother such that not only type and numbers of immune competent cells inthe tumor sample can be identified, but also their physiologicalstatus/activity and impact on tumor cells. Among other immune cells,especially contemplated immune competent cells include dendritic cells,CD4+T cells, CD8+T cells, cytotoxic T cells, NK cells, NKT cells, and M1and M2 macrophages. Additionally, while some types of cells can bevisually distinguished from other types of cells by their uniquemorphology, the inventors contemplate that tumor cells and/or varioustypes of immune cells among heterogeneous cell populations in the tumortissue can be visualized and detected by live labeling of the cells. Anysuitable labeling methods that can label the cells without substantiallyinterfering cell-cell interactions are contemplated. For example, sometumor cells and/or immune cells in the live tissue (or in thedisassociated cell culture) can be labeled with a quantum dot coupledwith a fluorescent dye (e.g., fluorescein isothiocyanate (FITC), etc.)and conjugated with peptide (e.g., single-chain variable fragment (scFv)or antibody fragment specific to a marker (e.g., antigens, receptorprotein, etc.) of a type of cell, etc.).

Preferably, the immune status of the tumor can be determined from themass of the tumor cell and/or the mass of the immune cells (and withthat the anticipated immune response of the patient to a tumor, can bereadily predicted by observing the cell mass of tumor cells and/orimmune cells (e.g., immune competent cells) within a sample. Theinventors contemplate that the standard (or threshold) for determiningthe immune status of the tumor may vary depending on the type of tumor,the patient's condition, and/or the patient's medical history. Forexample, the immune status of the tumor can be determined asimmune-sensitive or “hot” if the tumor cell proximal to the immune cellshows the mass that is at least 10%, at least 20%, at least 30%, atleast 40%, at least 50% less than other tumor cells, which do notproximally located to immune cells. For other example, the immune statusof the tumor can be determined as immune-sensitive or “hot” if theimmune cell proximal to the tumor cell shows the mass that is at least10%, at least 20%, at least 30%, at least 40%, at least 50% more thanother tumor cells, which do not proximally located to tumor cells.Conversely, the immune status of the tumor can be determined asimmune-resistant or “cold” if the tumor cell proximal to the immune cellshows the mass that is substantially same or even at least 10%, at least20%, at least 30%, at least 40%, at least 50% more than other tumorcells, which do not proximally located to immune cells. For otherexample, the immune status of the tumor can be determined asimmune-resistant or “cold”, if the immune cell proximal to the tumorcell shows the mass that is substantially same or even at least 10%, atleast 20%, at least 30%, at least 40%, at least 50% less than othertumor cells, which do not proximally located to tumor cells.

It is also contemplated that the immune status of the tumor can bedetermined by measuring collective or average mass of the tumor cells orimmune cells in the tumor tissue and comparing with the average mass ofnon-tumor cells or immune cells, respectively, in the non-diseasedtissue. For example, the mass of at least 20, at least 50, at least 100,or at least 300 tumor cells or immune cells in the tumor tissue, and themass of substantially same number of non-tumor cells (or the same organ,for example, liver of healthy individual, noncancerous portion of theliver of the patient, etc.) can be measured under LCI, and the averagemass of the tumor cells (or non-tumor cells) or immune cells can becalculated. The immune status of the tumor can be determined asimmune-sensitive or “hot” if average mass of the tumor cell is at least10%, at least 20%, at least 30%, at least 40%, at least 50% less thanother similar-sized non-tumor cells (known to have similar size withaverage size of such type of tumor cells, etc.), or at least 10%, atleast 20%, at least 30%, at least 40%, at least 50% less than othertumor cells present in different tumor mass in the same patient. Also,the immune status of the tumor can be determined as immune-sensitive or“hot” if average mass the immune cells in the sample is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50% more (larger) thanthe immune cells in the normal tissue (non-diseased tissue of thepatient, or tissue from the healthy individual, etc.). Conversely, Theimmune status of the tumor can be determined as immune-resistant or“cold” if average mass of the tumor cell is substantially same or evenat least 10%, at least 20%, at least 30%, at least 40%, at least 50%larger than other similar-sized non-tumor cells (known to have similarsize with average size of such type of tumor cells, etc.), or at least10%, at least 20%, at least 30%, at least 40%, at least 50% less thanother tumor cells present in different tumor mass in the same patient.Also, the immune status of the tumor can be determined asimmune-resistant or “cold” if average mass the immune cells in thesample is substantially same or even at least 10%, at least 20%, atleast 30%, at least 40%, at least 50% less (smaller) than the immunecells in the normal tissue (non-diseased tissue of the patient, ortissue from the healthy individual, etc.).

The inventors contemplate that the behavior and/or interaction of theimmune cell with the tumor cell in the tumor sample may not accuratelyreflect the in vivo behavior and/or interaction of the immune cell asthe condition of biopsy and/or acute processing of the tumor tissue maydiffer from the in vivo condition (e.g., temperature, excessive cellstress from the invasive tissue excision, etc.). Thus, in someembodiments, the mass of the tumor cells and/or immune cells from thetumor sample can be determined over a period of time after the tumortissue (or disassociated cells from the tumor cells) are placed on theex vivo culture condition. Any suitable time windows and/or duration foranalyzing cell mass in the tumor tissue are contemplated. For example,observation and analysis of cell mass under LCI can be performed in atleast 1 hour, at least 3 hours, at least 6 hours, at least 12 hours, atleast 24 hours, at least 3 days after the biopsy tissue was placed inthe ex vivo culture condition (either processed or unprocessed). Onceplaced under LCI, the observation and analysis of cell mass of tumorcells and/or immune cells can be performed at least at least 1 hour, atleast 3 hours, at least 6 hours, at least 12 hours continuously orperiodically (e.g., every 5 min, every 10 min, every 30 min, every 1hour, every 2 hours during the duration, etc.).

In some embodiments, where the LCI analysis is performed periodically onimmune cells and/or tumor cells, it is contemplated that the tissuesand/or cells can be placed under the LCI and the LCI isactivated/operated periodically. For example, the LCI can be turned on(light on) to measure the cell mass every 1 min, every 5 min, every 10min, every 30 min, every 1 hour, every 2 hours, while tissues and/orcells remains non-manipulated (e.g., not moved, untouched, etc.) duringthe measurements and in between the measurements. In other embodiments,the tissues and/or cells can be placed on the rotating/moving holder(e.g., tissue culture inserts), such that the tissues and/or cells canbe placed directly under the light of LCI for every 1 min, every 5 min,every 10 min, every 30 min, every 1 hour, every 2 hours for a durationof measurement and moves away from the light. In such embodiments, it ispreferred that a plurality of tissues and/or cells can be placed in adistinct location of the moving holder (e.g., multiple distinct tissueculture inserts on 6 well plate or petri dish, etc.) such that at leastone or more tissues/cells can be measured and analyzed while the othertissues/cells are in between two measurements.

Additionally, the inventors contemplate that the efficacy of a treatmentcan be tested real-time on the tumor tissue/cells under LCI such thatthe efficacy of the treatment in vivo can be predicted. In someembodiments, the tumor sample (tissue or cells) can be treated with oneor more treatment (e.g., antibodies, vaccines, checkpoint inhibitors,etc.), preferably after measuring the immune status of the tumor sample.For example, the tumor sample can be treated with one or more treatment(e.g., antibodies, vaccines, checkpoint inhibitors, etc.) and cell masschange of the tumor cell and/or immune cells can be measured real-timefor a duration of time (e.g., at least 1 hour, at least 3 hours, atleast 6 hours, etc.). The change of cell mass as a function of time(e.g., the shape of mass curve as a function of time if plotted on agraph, etc.) can provide a signature of the efficacy or metabolism oftreatment. For example, for treatment A, the cell mass change may occurearly (e.g., within 30 min) and be maintained for next 6 hours. Fortreatment B, the cell mass change may not occur until 6 hours after thetreatment. Based on such signature, the efficacy and metabolism of thetreatment can be similarly predicted in vivo such that the treatmentoptions can be modulated and selected.

Further, measurement of cell mass can be used to measure or predict themetastasis rate. Some tumor cells that are undergoing metastasis oftenhave altered cytoskeletal properties, in particular to be moredeformable and contractile. Thus, in one embodiment, the change of aplurality of tumor cells in the tumor sample can be measured such thatinformation of the ratio of the deformed cells in the tumor sampleand/or the degree of deformation as a function of time can be obtained.From at least one of the ratio of the deformed cells in the tumor sampleand/or the degree of deformation as a function of time, the metastasisrate of the tumor cells in the tumor sample can be measured and themetastasis rate of tumor cells of the tumor in vivo can be predicted.

Alternatively, the inventors also contemplate that tumor cell-immunecell interaction conditions can be artificially made with morehomogeneous cell populations by using the artificial tumor massgenerated from the tumor cells of the tumor tissue sample. In oneembodiment, various types of endogenous, naive immune cells from thetumor tissue sample (or circulating cells, or cells from a lymph node)can be isolated and separated by cell types (e.g., CD4+T cells, CD8+Tcells, B cells, by Fluorescence-activated cell sorting (FACS) or usingantibodies specific to a marker of specific immune cells (Vα24-Jα18 forhuman NKT cells, etc.), etc.). In addition, it is also contemplated thatsome immune cells can be isolated from the tumor tissue and can befurther expanded ex vivo to increase the number of the cells for furtherexperiments and analysis. For example, NK cells or NKT cells can beplaced in a cell culture media (e.g., AIMV® medium, RPMI1640® etc.) thatincludes one or more activating conditions. The activating conditionsmay include addition of any molecules that can stimulate NK or NKTgrowth, induce cell division of NK or NKT, and/or stimulate cytokinerelease from NK or NKT that can further expand NK or NKT cells. It iscontemplated that the activating conditions may vary depending on thetiming of the ex vivo expansion and activation.

For example, NK cell expansion can be performed using various activatingmolecules added in the culture media including cytokines (e.g., IL-2,IL-15, etc.), monoclonal antibodies (e.g., murine monoclonal antibodyagainst CD3 (OKT3TM), etc.), or using cell-to-cell interaction withactivating cells (e.g., K562 cells, a cell line derived from a patientwith myeloid blast crisis of chronic myelogenous leukemia and bearingthe BCR-ABL1 translocation, etc.)

With respect to NKT cells, ex vivo expansion and activation of NKT cellscan be performed using the activator of endogenous NKT T cell receptoror antibodies against the components of the endogenous NKT T cellreceptor, before the endogenous NKT T cells are removed by knock-in ofrecombinant nucleic acid. However, after the endogenous NKT T cells areremoved by knock-in of recombinant nucleic acid, it is contemplated thatthe activator of endogenous NKT T cell receptor or antibodies againstthe components of the endogenous NKT T cell receptor may not be used foreffective ex vivo expansion and activation.

Thus, the activating molecules may include T cell receptor antibodies(e.g., anti-CD2, anti-CD3, anti-CD28, α-TCR-V α24+antibodies, preferablyimmobilized on beads, etc.), a glycolipid (e.g., α-GlcCer, α-ManCer,GD3, etc.), a glycolipid coupled with CD1 (e.g., CD1d, etc.) if the exvivo expansion and activation is performed before the recombinantnucleic acid is introduced into the NKT cells. After the recombinantnucleic acid is introduced into the NKT cells, the activating moleculesmay include one or more cytokines (e.g., IL-2, IL-5, IL-7, IL-8, IL-12,IL-12, IL-15, IL-18, and IL-21, preferably human recombinant IL-2, IL-5,IL-7, IL-8, IL-12, IL-12, IL-15, IL-18, and IL-21, etc.) in anydesirable concentration (e.g., at least 10 U/ml, at least 50 U/ml, atleast 100 U/ml), etc. In some embodiments, the activation conditions mayinclude culturing the isolated and enriched NKT cells with autologous orallogeneic peripheral blood mononuclear cells (PBMC) feeder cells.

The isolated immune cells can further be placed close to the artificialtumor (e.g., in the same culture dish, within less than 5 cm distance,less than 3 cm distances, less than 1 cm distance, less than 0.5 cmdistance, etc.), and co-incubate with the artificial tumor mass for atleast 3 hours, at least 6 hours, at least 12 hours, at least 24 hours,at least 3 days such that the immune cells can infiltrate the artificialtumor mass and elicit the immune response against the tumor cells. Insome embodiments, a plurality of artificial tumor mass can be generatedfrom a single biopsy tumor tissue, and each of the artificial tumor masscan be contacted with different types of isolated immune cells (CD4+Tcells, CD8+T cells, NK cells, NKT cells, macrophage, etc.) or acombination of isolated immune cells (e.g., at least two of immunecompetent cells in a ratio of at least 1:1, at least 1:2, at least 1:3,at least 1:5, etc.).

In some embodiment, the tumor samples (acute slice, cultured slice,disassociated cells, derived from the biopsy tumor tissue) and/orartificial tumor can be contacted (or at least co-placed with)genetically modified or genetically engineered immune cells. Forexample, the artificial tumor can be contacted NK92 cells andderivatives thereof (e.g., aNK cells, haNK cells, taNK cells, allcommercially available from NantKwest, 9920 Jefferson Blvd. Culver City,Calif. 90232). For other example, the tumor samples and/or theartificial tumor can be contacted genetically engineered T cells, NKcells, or NKT cells expressing a recombinant chimeric antigenic receptor(CAR) that specifically bind tumor antigen or neoepitope expressed bythe tumor cells in the biopsy tumor tissue. For still other example, thetumor samples and/or the artificial tumor can be contacted ex vivoactivated cytotoxic immune cells (e.g., ex vivo expanded and/oractivated NK cells or NKT cells with cytokines, etc.). With that, theinventors contemplate that the immune status of the tumor to the cancervaccine or modified immune cells can be determined such that theanticipated effect of the cancer vaccine or modified immune cellsagainst the tumor cells can be predicted. In such example, it ispreferred that the tumor-associated antigens or neoepitopes that aretumor-specific and/or patient-specific are identified and one or morenucleic acid constructs encoding CAR specific to those tumor-associatedantigens or neoepitopes. Typically, the tumor associated antigens and/orneoepitopes (which are typically patient-specific and tumor-specific)can be identified from the omics data obtained from the cancer tissue ofthe patient or normal tissue (of the patient or a healthy individual),respectively. Omics data typically includes information related togenomics, transcriptomics, and proteomics.

The artificial tumor mass contacted (or at least co-placed with) theisolated immune cells and the artificial tumor mass uncontacted theisolated immune cells then can be further observed to determine the cellmass of the tumor cells and immune cells in the tumor mass. In suchembodiment, the immune status of the tumor cells in the tumor (asreflected in artificial tumor mass) can be determined asimmune-sensitive or “hot” if average mass of the tumor cell in theartificial tumor contacting the immune cells is at least 10%, at least20%, at least 30%, at least 40%, at least 50% less than average mass ofthe tumor cell in the artificial tumor without contacting the immunecells. Also, the immune status of the tumor can be determined asimmune-sensitive or “hot” if average mass the immune cells in theartificial tumor contacting the immune cells is at least 10%, at least20%, at least 30%, at least 40%, at least 50% more (larger) than theimmune cells before or without contacting the artificial tumor mass.Conversely, the immune status of the tumor cells in the tumor (asreflected in artificial tumor mass) can be determined asimmune-resistant or “cold” if average mass of the tumor cell in theartificial tumor contacting the immune cells is substantially same oreven at least 10%, at least 20%, at least 30%, at least 40%, at least50% larger (more) than average mass of the tumor cell in the artificialtumor without contacting the immune cells. Also, the immune status ofthe tumor can be determined as immune- resistant or “cold” if averagemass the immune cells in the artificial tumor contacting the immunecells is substantially same or even at least 10%, at least 20%, at least30%, at least 40%, at least 50% less (smaller) than the immune cellsbefore or without contacting the artificial tumor mass.

Additionally, the inventors contemplate that the immune status of thetumor can be determined by considering other information on the tumorcells and/or immune cells in addition to the cell mass of the tumor cellor the immune cell. In one embodiment, ratios among different types ofimmune cells in an area of the tumor tissue (e.g., per 1 mm², per 10mm², per 100 mm², etc.) can be determined and used as an indicator ofthe immune status as one or more different types of immune competentcells may serve as a proxy indicator for immunogenicity of the tumor.For example, where the tumor is infiltrated with M2 macrophages and isrelatively poor in NK cells and CD8+T cells, the tumor of the patientmay be deemed immunologically “cold”. On the other hand, where the tumoris relatively rich in CD4+/CD8+T cells and NK cells, the tumor of thepatient may be immunologically “hot”.

In another embodiment, the morphologies (e.g., shape, height, etc.) ofthe tumor cells and/or immune cells (of the same type), degree of cellto cell adhesion, or cell-extracellular matrix adhesion of the tumorcells and/or immune cells, or relative locations of tumor cells andimmune cells in the tumor tissue can be observed and analyzed to deriveimmune status information. For example, where the immune cells,especially cytotoxic immune cells, in the tumor tissue are not locatedin vicinity of the tumor cells, such relative location information mayindicate that the tumor may be deemed immunologically “cold” either byprohibiting the infiltration of the cytotoxic immune cells to the tumorto have a contact to the tumor cells or by actively removing theinfiltrated cytotoxic immune cells from the tumor microenvironment.

Some immunologically “cold” tumor may be induced to be at leastimmunologically neutral or even “hot” if the tumor microenvironment isoptimized. Thus, inventors further contemplate that the tumor sampledetermined to be “cold” can be further treated with one or moreco-stimulatory molecules, an immune stimulatory cytokine, and/or aprotein that interferes with or down-regulates checkpoint inhibition. Insome embodiments, can be analyzed under LCI and upon determination ofits immune status as “cold”, the tumor sample can be treated with one ormore co-stimulatory molecules, an immune stimulatory cytokine, and/or aprotein that interferes with or down-regulates checkpoint inhibition forat least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours,at least 3 days, at least 7 days before determining the cell mass oftumor cells and/or immune cells under LCI again. In other embodiments,the tumor sample can be divided into at least two badges (e.g., twoacute slices, two separate cell cultures from the same biopsy tumortissue, maintained in the same condition, etc.). In these embodiments,one batch of the tumor sample can be used without treatment to determinethe mass of tumor cells and/or immune cells, and another badge of thetumor sample can be used after treatment with co-stimulatory moleculesor their ligands, an immune stimulatory cytokine, and/or a protein thatinterferes with or down-regulates checkpoint inhibition to determine themass of tumor cells and/or immune cells.

Suitable co-stimulatory molecules include, but not limited to, CD80,CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4,CD70, OX40L,4-1BBL, while other stimulatory molecules with less defined (orunderstood) mechanism of action include GITR-L, TIM-3, TIM-4, CD48,CD58, TL1A, ICAM-1, LFA3, and members of the SLAM family. In addition,any suitable types of cytokines to boost the immune response arecontemplated. Especially preferred cytokines and cytokine analogsinclude IL-2, IL-15, and IL-15 superagonist (ALT-803), IL-21, IPS1, andLMP1.

With respect to a protein that interferes with or down-regulatescheckpoint inhibition, it is contemplated any suitable peptide ligandsthat bind to a checkpoint receptor are contemplated. Most typically,binding will inhibit or at least reduce signaling via the receptor, andparticularly contemplated receptors include CTLA-4 (especially forCD8⁺cells), PD-1 (especially for CD4⁺cells), TIM1 receptor, 2B4, andCD160. For example, suitable peptide binders can include antibodyfragments and especially scFv, but also small molecule peptide ligands(e.g., isolated via RNA display or phage panning) that specifically bindto the receptors (e.g., ipilimumab, nivolumab, etc.).

In addition, it is contemplated that where the tumor sample determinedto be “cold”, some “cold” tumor samples may be transformed to “hot” bytreating the tumor cells with stress molecules that can induce the tumorcells to be immunogenic. For example, tumor cells can be treated with acontinuous exposure or metronomic exposure (e.g., using one or morechemotherapeutic drugs at relatively low dose) or other stressconditions such as hypoxia, local heat shock treatment (e.g., at 42degree celcius for 1 min, for 3 min, for 5 min, etc.), exposure totoxins and/or mechanical damage (e.g., partial surgical removal ofcancer tissue, etc.). In such embodiment, responsiveness of a tumor(reflected by changes in mass of tumor cells or immune cells) may beevaluated over multiple LCI measurements to identify the optimal stressconditions to convert the immune status of the tumor.

Moreover, it is contemplated that where the tumor sample determined tobe “cold”, the “cold” tumor samples may be transformed to “hot” byinducing a condition in the tumor to attract more immune competentcells. For example, the tumor tissue may be pre-treated with chemokinesthat may be effective in chemo-attraction of immune competent cells, andespecially CD8+ and CD4+T cells, for at least 30 min, at least 1 hour,at least 3 hours, at least 6 hours, at least 12 hours beforere-evaluating responsiveness of a tumor (reflected by changes in mass oftumor cells or immune cells) over multiple LCI measurements.

The inventors contemplate that the immune response against tumor bypatient's own immune system or boosted by immunotherapy can be predictedbased on the determined immune status. As used herein, the immuneresponse that is predicted based on the immune status refers aeffectiveness or responsiveness of a given immunotherapy against thetumor or a likelihood of success of the immunotherapy against the tumor(e.g., by decreasing the tumor size at least 20%, at least 30%, at least40%, at least 50%, etc., by suppressing the metastasis of the tumor,etc.). Without wishing to be bound to any specific theory, theimmunologically “hot” tumors are more likely to respond toindividualized immunotherapy (e.g., personalized cancer vaccine, etc.)such that the individualized immunotherapy or other types ofimmunotherapy can be highly recommended as a treatment option againstthe tumor. Conversely, when the tumor is determined to beimmunologically “cold”, those tumors are less likely to respond toindividualized immunotherapy (e.g., personalized cancer vaccine, etc.)such that the individualized immunotherapy or other types ofimmunotherapy may not be the first prioritized option to treat thetumor. In this case, some additional or other types of treatment optionscan be provided. For example, where the tumor determined to beimmunologically “cold”, yet the tumor is conditionally “hot” when theimmune stimulatory molecule is pre-treated, the treatment option forco-treatment or sequential treatment of one of more immune stimulatorymolecules with personalized cancer vaccine can be recommended.

The inventors further contemplate that the metastasis rate orpossibility of metastasis originated from the tumor can be predictedbased on the determined immune status. Without wishing to be bound toany specific theory, the immunologically “hot” tumors are less likely tometastasize to other part of the body such that metastasis rate orpossibility of metastasis from the tumor is relatively low. Conversely,when the tumor is determined to be immunologically “cold”, those tumorsare more likely to metastasize to other part of the body such thatmetastasis rate or possibility of metastasis from the tumor isrelatively high.

Optionally, the tumor tissue and/or sample that was used to evaluate theimmune status of the tumor or the tumor tissues can be further placedfor additional molecular and cellular analysis. For example, where thetumor tissue is obtained from one of the metastasized tumors and thetumor tissue is determined immunologically hot or cold, omics data canbe obtained from the tumor cells in the tumor tissue to identify any keymolecules and/or mutations that contributes the immune status of thetumor tissue. In some embodiments, a tumor cell that showed response ornonresponse to the immune cell under LCI can be picked up using amicropipette and the genetic materials (e.g., DNA, RNA, etc.) can beretrieved from the single tumor cell. In those embodiments, themolecular analysis and omics data analysis can be performed in asingle-cell level.

For other example, the tumor tissue and/or sample that was used toevaluate the immune status of the tumor or the tumor tissues can befurther processed (e.g., freezing, fixing with formalin-fixedparaffin-embedded (FFPE) technique, fixing with paraformaldehyde (PFA),etc.) so that the tissue can be analyzed using immunohistochemicalassays or biochemical assays (e.g., western blotting, northern blotting,etc.) to determine the cell types (e.g., using cell-specific markers,etc.), distribution of different types of cells in the tumor tissuerelative to the tumor cells (e.g., location and accumulation of MDSC,Treg, proximity of different types of immune cells to the tumor cells,etc.), or cellular and molecular changes of the immune cells and/ortumor cells (e.g., expression level of receptors in different types ofimmune cells, heterogeneity of tumor cells, etc.) in the immunologicallyhot or cold tumor tissues.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Unless the context dictates the contrary,all ranges set forth herein should be interpreted as being inclusive oftheir endpoints, and open-ended ranges should be interpreted to includecommercially practical values. Similarly, all lists of values should beconsidered as inclusive of intermediate values unless the contextindicates the contrary.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

1. A method of predicting an immune response against a tumor in apatient having the tumor, comprising: obtaining, prior to animmunotherapeutic treatment, a tumor sample from a patient; determining,within the tumor sample, at least one of a mass of a tumor cell and amass of an immune competent cell using live cell interferometry; usingthe at least one of the mass of the tumor cell and the mass of theimmune competent cell to determine an immune status of the tumor sample;and using the immune status to predict the immune response against thetumor in the patient.
 2. The method of claim 1, further comprising astep of identifying a type of the immune competent cell.
 3. The methodof claim 1, wherein the determining the at least one of the mass of thetumor cell and the mass of the immune competent cell comprises measuringchanges over a period of time in the at least one of the mass of thetumor cell and the mass of the immune competent cell.
 4. The method ofclaim 1, wherein the mass of the tumor cell is compared with an averagemass of other tumor cells in the tumor sample.
 5. The method of claim 1,wherein the mass of the immune competent cell is compared with anaverage mass of immune competent cells in the tumor sample.
 6. Themethod of claim 1, wherein the tumor cell is located proximal to theimmune competent cell.
 7. The method of claim 1, further comprisingcontacting the tumor sample with an immune stimulatory cytokine, acheckpoint inhibitor, or an immunotherapeutic agent.
 8. (canceled) 9.(canceled)
 10. The method of claim 7, further comprising comparing theat least one of a mass of a tumor cell and a mass of an immune competentcell before and after contacting the tumor sample with theimmunotherapeutic agent.
 11. The method of claim 1, wherein the immunestatus is determined to be hot when the mass of the tumor cell is atleast 20 percent larger than other tumor cells in the tumor sample. 12.The method of claim 1, wherein the immune status is determined to be hotwhen the mass of the immune competent cell is at least 20 percent largerthan other immune competent cells in the tumor sample.
 13. (canceled)14. The method of claim 1, wherein the tumor sample is a portion of anartificial tumor that is generated from a tumor biopsy of the patient.15. The method of claim 1, wherein the immune competent cell is labeledwith a marker.
 16. The method of claim 1, wherein the mass of the tumorcell is determined by comparing optical thickness of the tumor cell andoptical thickness of a control cell.
 17. The method of claim 1, furthercomprising fixing the tumor cell and the immune competent cell in thetumor sample and identifying a type of at least one of the tumor celland the immune competent cell by labeling with a marker.
 18. The methodof claim 1, wherein the tumor cell and the immune competent cell isdisassociated and placed in cell culture medium.
 19. The method of claim1, wherein the tumor cell and the immune competent cell is in theacutely sliced tissue.
 20. The method of claim 1, wherein the tumor celland the immune competent cell is in the cultured sliced tissue.
 21. Themethod of claim 1, further comprising identifying presence of aneoepitope on the tumor cell, wherein the neoepitope is specific for thepatient and the tumor.
 22. The method of claim 1, wherein the immuneresponse is predicted positive when the immune status is determined tobe hot.
 23. The method of claim 1, further comprising providing analternative option for the immunotherapeutic treatment when the immunestatus is determined to be cold.